Clare R. Sander

Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans

Protective immunity against Mycobacterium tuberculosis depends on the generation of a TH1-type cellular immune response, characterized by the secretion of interferon-γ (IFN-γ) from antigen-specific T cells. The induction of potent cellular immune responses by vaccination in humans has proven difficult. Recombinant viral vectors, especially poxviruses and adenoviruses, are particularly effective at boosting previously primed CD4+ and CD8+ T-cell responses against a number of intracellular pathogens in animal studies. In the first phase 1 study of any candidate subunit vaccine against tuberculosis, recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) was found to induce high levels of antigen-specific IFN-γ-secreting T cells when used alone in bacille Calmette-Guérin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5–38 years previously with BCG, substantially higher levels of antigen-specific IFN-γ-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5–30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas.

Multifunctional, High-Level Cytokine-Producing Th1 Cells in the Lung, but Not Spleen, Correlate with Protection against Mycobacterium tuberculosis Aerosol Challenge in Mice

Boosting bacillus Calmette-Guérin (BCG)-primed mice with a recombinant adenovirus expressing Mycobacterium tuberculosis Ag 85A by different administration routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to Ag 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast, intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to Ag 85A and an increased response to purified protein derivative. This effect is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensity and integrated median fluorescence intensity for IFN-γ, IL-2, and TNF. In contrast, mice immunized with BCG alone have few Ag-specific cells in the lung and a low proportion of multifunctional cells, although individual cells have high median fluorescence intensity. Successful immunization regimes appear to induce Ag-specific cells with abundant intracellular cytokine staining.

Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional mycobacterium tuberculosis-specific CD4+ memory T lymphocyte populations

In the search for effective vaccines against intracellular pathogens such as HIV, tuberculosis and malaria, recombinant viral vectors are increasingly being used to boost previously primed T cell responses. Published data have shown prime‐boost vaccination with BCG‐MVA85A (modified vaccinia virus Ankara expressing antigen 85A) to be highly immunogenic in humans as measured by ex vivo IFN‐γ ELISPOT. Here, we used polychromatic flow cytometry to investigate the phenotypic and functional profile of these vaccine‐induced Mycobacterium tuberculosis (M.tb) antigen 85A‐specific responses in greater detail. Promisingly, antigen 85A‐specific CD4+ T cells were found to be highly polyfunctional, producing IFN‐γ, TNF‐α, IL‐2 and MIP‐1β. Surface staining showed the responding CD4+ T cells to be relatively immature (CD45RO+ CD27intCD57–); this observation was supported by the robust proliferative responses observed following antigenic stimulation. Furthermore, these phenotypic and functional properties were independent of clonotypic composition and epitope specificity, which was maintained through the different phases of the vaccine‐induced immune response. Overall, these data strongly support the use of MVA85A in humans as a boosting agent to expand polyfunctional M.tb‐specific CD4+ T cells capable of significant secondary responses.

Safety and Immunogenicity of a New Tuberculosis Vaccine, MVA85A, in Mycobacterium tuberculosis–infected Individuals

RATIONALE An effective new tuberculosis (TB) vaccine regimen must be safe in individuals with latent TB infection (LTBI) and is a priority for global health care. OBJECTIVES To evaluate the safety and immunogenicity of a leading new TB vaccine, recombinant Modified Vaccinia Ankara expressing Antigen 85A (MVA85A) in individuals with LTBI. METHODS An open-label, phase I trial of MVA85A was performed in 12 subjects with LTBI recruited from TB contact clinics in Oxford and London or by poster advertisements in Oxford hospitals. Patients were assessed clinically and had blood samples drawn for immunological analysis over a 52-week period after vaccination with MVA85A. Thoracic computed tomography scans were performed at baseline and at 10 weeks after vaccination. Safety of MVA85A was assessed by clinical, radiological, and inflammatory markers. The immunogenicity of MVA85A was assessed by IFNgamma and IL-2 ELISpot assays and FACS. MEASUREMENTS AND MAIN RESULTS MVA85A was safe in subjects with LTBI, with comparable adverse events to previous trials of MVA85A. There were no clinically significant changes in inflammatory markers or thoracic computed tomography scans after vaccination. MVA85A induced a strong antigen-specific IFN-gamma and IL-2 response that was durable for 52 weeks. The magnitude of IFN-gamma response was comparable to previous trials of MVA85A in bacillus Calmette-Guérin-vaccinated individuals. Antigen 85A-specific polyfunctional CD4(+) T cells were detectable prior to vaccination with statistically significant increases in cell numbers after vaccination. CONCLUSIONS MVA85A is safe and highly immunogenic in individuals with LTBI. These results will facilitate further trials in TB-endemic areas. Clinical trial registered with www.clinicaltrials.gov (NCT00456183).

Safety and immunogenicity of boosting BCG vaccinated subjects with BCG: Comparison with boosting with a new TB vaccine, MVA85A

Objectives To investigate the safety and immunogenicity of a booster BCG vaccination delivered intradermally in healthy, BCG vaccinated subjects and to compare with a previous clinical trial where BCG vaccinated subjects were boosted with a new TB vaccine, MVA85A. Design Phase I open label observational trial, in the UK. Healthy, HIV-negative, BCG vaccinated adults were recruited and vaccinated with BCG. The primary outcome was safety; the secondary outcome was cellular immune responses to antigen 85, overlapping peptides of antigen 85A and tuberculin purified protein derivative (PPD) detected by ex vivo interferon-gamma (IFN-γ) ELISpot assay and flow cytometry. Results and Conclusions BCG revaccination (BCG-BCG) was well tolerated, and boosting of pre-existing PPD-specific T cell responses was observed. However, when these results were compared with data from a previous clinical trial, where BCG was boosted with MVA85A (BCG-MVA85A), MVA85A induced significantly higher levels (>2-fold) of antigen 85-specific CD4+ T cells (both antigen and peptide pool responses) than boosting with BCG, up to 52 weeks post-vaccination (p = 0.009). To identify antigen 85A-specific CD8+ T cells that were not detectable by ex vivo ELISpot and flow cytometry, dendritic cells (DC) were used to amplify CD8+ T cells from PBMC samples. We observed low, but detectable levels of antigen 85A-specific CD8+ T cells producing IFNγ (1.5% of total CD8 population) in the BCG primed subjects after BCG boosting in 1 (20%) of 5 subjects. In contrast, in BCG-MVA85A vaccinated subjects, high levels of antigen 85A-specific CD8+ T cells (up to 14% total CD8 population) were observed after boosting with MVA85A, in 4 (50%) of 8 subjects evaluated. In conclusion, revaccination with BCG resulted in modest boosting of pre-existing immune responses to PPD and antigen 85, but vaccination with BCG-MVA85A induced a significantly higher response to antigen 85 and generated a higher frequency of antigen 85A-specific CD8+ T cells. Trial Registration ClinicalTrials.gov NCT00654316 NCT00427830

Boosting BCG with Recombinant Modified Vaccinia Ankara Expressing Antigen 85A: Different Boosting Intervals and Implications for Efficacy Trials

Objectives To investigate the safety and immunogenicity of boosting BCG with modified vaccinia Ankara expressing antigen 85A (MVA85A), shortly after BCG vaccination, and to compare this first with the immunogenicity of BCG vaccination alone and second with a previous clinical trial where MVA85A was administered more than 10 years after BCG vaccination. Design There are two clinical trials reported here: a Phase I observational trial with MVA85A; and a Phase IV observational trial with BCG. These clinical trials were all conducted in the UK in healthy, HIV negative, BCG naïve adults. Subjects were vaccinated with BCG alone; or BCG and then subsequently boosted with MVA85A four weeks later (short interval). The outcome measures, safety and immunogenicity, were monitored for six months. The immunogenicity results from this short interval BCG prime–MVA85A boost trial were compared first with the BCG alone trial and second with a previous clinical trial where MVA85A vaccination was administered many years after vaccination with BCG. Results MVA85A was safe and highly immunogenic when administered to subjects who had recently received BCG vaccination. When the short interval trial data presented here were compared with the previous long interval trial data, there were no significant differences in the magnitude of immune responses generated when MVA85A was administered shortly after, or many years after BCG vaccination. Conclusions The clinical trial data presented here provides further evidence of the ability of MVA85A to boost BCG primed immune responses. This boosting potential is not influenced by the time interval between prior BCG vaccination and boosting with MVA85A. These findings have important implications for the design of efficacy trials with MVA85A. Boosting BCG induced anti-mycobacterial immunity in either infancy or adolescence are both potential applications for this vaccine, given the immunological data presented here. Trial Registration ClinicalTrials.gov NCT00427453 (short boosting interval), NCT00427830 (long boosting interval), NCT00480714 (BCG alone)

Investigating the Induction of Vaccine-Induced Th17 and Regulatory T Cells in Healthy, Mycobacterium bovis BCG-Immunized Adults Vaccinated with a New Tuberculosis Vaccine, MVA85A

ABSTRACT Tuberculosis (TB) remains a threat to global health. While advances in diagnostics and treatment are crucial to the containment of the epidemic, it is likely that elimination of the disease can only be achieved through vaccination. Vaccine-induced protection from Mycobacterium tuberculosis is dependent, at least in part, on a robust Th1 response, yet little is known of the ability of TB vaccines to induce other T-cell subsets which may influence vaccine efficacy. Interleukin-17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells which has been associated with both immune pathology and protection against infectious disease. Following vaccination with MVA85A, a viral vector vaccine aimed at enhancing immune responses to M. tuberculosis, antigen-specific IL-17A-producing T cells were induced in the peripheral blood of healthy volunteers. These T cells are detected later than gamma interferon (IFN-γ)-secreting T cells and are of a low magnitude. Preexisting immune responses to mycobacterial antigens were associated with higher CD4+ CD25hi CD39+ T-cell levels in the periphery and a reduced capacity to produce IL-17A following immunization. These data highlight the intricate balance of effector and regulatory immune responses induced by vaccination and that preexisting immunity to mycobacterial antigens may affect the composition of vaccine-induced T-cell subsets.

Vaccine Platform for Prevention of Tuberculosis and Mother-to-Child Transmission of Human Immunodeficiency Virus Type 1 through Breastfeeding

ABSTRACT Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born to human immunodeficiency virus type 1 (HIV-1)-positive mothers are at high risk of acquiring HIV-1 infection through breastfeeding in the first weeks of their lives. Thus, the development of a vaccine which would protect newborns against both of these major global killers is a logical yet highly scientifically, ethically, and practically challenging aim. Here, a recombinant lysine auxotroph of Mycobacterium bovis bacillus Calmette-Guérin (BCG), a BCG strain that is safer than those currently used and expresses an African HIV-1 clade-derived immunogen, was generated and shown to be stable and to induce durable, high-quality HIV-1-specific CD4+- and CD8+-T-cell responses. Furthermore, when the recombinant BCG vaccine was used in a priming-boosting regimen with heterologous components, the HIV-1-specific responses provided protection against surrogate virus challenge, and the recombinant BCG vaccine alone protected against aerosol challenge with M. tuberculosis. Thus, inserting an HIV-1-derived immunogen into the scheduled BCG vaccine delivered at or soon after birth may prime HIV-1-specific responses, which can be boosted by natural exposure to HIV-1 in the breast milk and/or by a heterologous vaccine such as recombinant modified vaccinia virus Ankara delivering the same immunogen, and decrease mother-to-child transmission of HIV-1 during breastfeeding.

A comparison of IFNγ detection methods used in tuberculosis vaccine trials

Interferon gamma (IFNgamma) is a critical component of the pro-inflammatory immune response that provides protection against Mycobacterium tuberculosis. In the absence of an immunological correlate of protection, antigen-specific production of IFNgamma is a commonly used marker of a protective immune response. To facilitate the evaluation of tuberculosis candidate vaccines three different IFNgamma detection methods were compared. The cultured whole blood ELISA, ex vivo IFNgamma ELISpot and whole blood ex vivo intracellular cytokine staining (ICS) assays were performed head-to-head during a Phase I clinical trial using the candidate vaccine MVA85A. Whilst all three assays detected significant increases in IFNgamma production immediately following vaccination, distinctions between the assays were apparent. Higher baseline IFNgamma responses were detected using the cultured whole blood ELISA, whereas the ex vivo ELISpot assay was the most sensitive in detecting long-term (52 weeks) post-vaccination responses. The whole blood ex vivo ICS assay provided novel information by dissecting the IFNgamma response into responding CD4, CD8 and gamma/delta T cell subsets. Future tuberculosis vaccine trials and immunology studies should ideally include a combination of ex vivo and cultured assays to ensure a thorough and multifaceted evaluation of the immune response is achieved.

A Phase I study evaluating the safety and immunogenicity of MVA85A, a candidate TB vaccine, in HIV-infected adults

Objectives Control of the tuberculosis (TB) epidemic is a global health priority and one that is likely to be achieved only through vaccination. The critical overlap with the HIV epidemic requires any effective TB vaccine regimen to be safe in individuals who are infected with HIV. The objectives of this clinical trial were to evaluate the safety and immunogenicity of a leading candidate TB vaccine, MVA85A, in healthy, HIV-infected adults. Design This was an open-label Phase I trial, performed in 20 healthy HIV-infected, antiretroviral-naïve subjects. Two different doses of MVA85A were each evaluated as a single immunisation in 10 subjects, with 24 weeks of follow-up. The safety of MVA85A was assessed by clinical and laboratory markers, including regular CD4 counts and HIV RNA load measurements. Vaccine immunogenicity was assessed by ex vivo interferon γ (IFN-γ) ELISpot assays and flow-cytometric analysis. Results MVA85A was safe in subjects with HIV infection, with an adverse-event profile comparable with historical data from previous trials in HIV-uninfected subjects. There were no clinically significant vaccine-related changes in CD4 count or HIV RNA load in any subjects, and no evidence from qPCR analyses to indicate that MVA85A vaccination leads to widespread preferential infection of vaccine-induced CD4 T cell populations. Both doses of MVA85A induced an antigen-specific IFN-γ response that was durable for 24 weeks, although of a lesser magnitude compared with historical data from HIV-uninfected subjects. The functional quality of the vaccine-induced T cell response in HIV-infected subjects was remarkably comparable with that observed in healthy HIV-uninfected controls, but less durable. Conclusion MVA85A is safe and immunogenic in healthy adults infected with HIV. Further safety and efficacy evaluation of this candidate vaccine in TB- and HIV-endemic areas is merited.